China Animal Husbandry and Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (5): 1966-1976.doi: 10.16431/j.cnki.1671-7236.2025.05.003

• Biotechnology • Previous Articles    

Establishment of ST Cell Lines with WIP1 Gene g.37536832 C＞A Mutation Using the CRISPR/Cas9 Gene Editing System

WANG Nan1,2, DU Weiwei3, WANG Wanjie1, WANG Yue1,4, YUAN Maosha1, NIE Yuxin1, SUN Yaru1, LIU Zhiguo1, WU Tianwen1, MU Yulian1   

  1. 1. Institute of Animal Science of Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China;
    3. Xinjiang Taiyu Breeding Pig Co., Ltd., Wujiaqu 831300, China;
    4. School of Life Science and Engineering, Foshan University, Foshan 528225, China
  • Received:2024-11-04 Published:2025-04-27

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Abstract: 【Objective】 The purpose of this experiment was to establish the wild-type p53-induced phosphatase 1 (WIP1) gene g.37536832 C＞A mutation swine testis (ST) cell line which was significantly associated with sperm motility by using CRISPR/Cas9 gene editing technology,and laid a solid foundation for further research on the relationship between the WIP1 gene and the semen quality traits of boar semen at the cellular level.【Method】 Three single guide RNA (sgRNA) were designed near the g.37536832 C＞A locus of porcine WIP1 gene using CRISPOR online website and connected to pX458-GFP vector.The activities of three sgRNA vectors were detected by T7E1 enzymatic cleavage assay,and the sgRNA with higher efficiency was selected.Donor vector was constructed and transfected into ST cells together with sgRNA vector.Positive cells expressing green fluorescent protein (GFP) were enriched and monoclonal cells were screened using flow cytometry,and the genotypes of the selected monoclonal cells were identified. 【Result】 Plasmid sequencing results showed that three sgRNAs were successfully ligated into the pX458-GFP vector.The results of T7E1 enzyme digestion showed that pX458-GFP-sgRNA1 and pX458-GFP-sgRNA2 transfected plasmids produced cleavage,and the efficiency was 24% and 27%,respectively,while no cleavage was detected in pX458-GFP-sgRNA3.Therefore,pX458-GFP-sgRNA2 plasmid was selected for the experiment.Donor vector was successfully constructed,and pX458-GFP-sgRNA2 and Donor vector were transfected into ST cells.Genotype identification showed that 205 of the 269 monoclones obtained had gene editing,and the gene editing efficiency was 76%.Among them,9 clones were ST cells with homozygous mutation at g.37536832 C>A of WIP1 gene,and the precise mutation efficiency was 3%.【Conclusion】 In this study,the ST cell line with the mutation of g.37536832 C＞A locus of WIP1 gene was successfully obtained using CRISPR/Cas9 gene editing technology,which provided a favourable cell model for further study of the effect of WIP1 gene on the semen quality of boars.

Key words: CRISPR/Cas9; WIP1 gene; ST cell line

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